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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.34 n.1 Santiago  2001 

Participation of the phosphoinositide metabolism in the
hypersensitive response of Citrus limon against
Alternaria alternata


1 Laboratorio de Bioquímica, Facultad Ciencias de la Salud, Universidad Nacional Andrés Bello.
2 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas,
Universidad de Chile


Lemon seedlings inoculated with Alternaria alternata develop a hypersensitive response (HR) that includes the induction of Phenylalanine ammonia-lyase (PAL, E. C. and the synthesis of scoparone. The signal transduction pathway involved in the development of this response is unknown. We used several inhibitors of the Phosphoinositide (PI) animal system to study a possible role of Inositol-1,4,5-triphosphate (IP3 ) in the transduction of the fungal conidia signal in Citrus limon. The HR was only partially inhibited by EGTA, suggesting that not only external but internal calcium as well are necessary for a complete development of the HR. In this plant system, Alternaria alternata induced an early accumulation of the second messenger IP3. When lemon seedlings were watered long term with LiCl, an inhibitor of the phosphoinositide cycle, the IP3 production was reduced, and the LiCl-watered plants could neither induce PAL nor synthesize scoparone in response to fungal conidia. Furthermore, neomycin, a Phospholipase C (PLC, E. C. inhibitor, also inhibited PAL induction and scoparone synthesis in response to A. alternata. These results suggest that IP3 could be involved in the signal transduction pathway for the development of the HR of Citrus limon against A. alternata.

Key terms: Citrus limon, IP3, phosphoinositides, Rutaceae, signal transduction


Plant defense against phytopathogen attack is a complex process. It depends on the plant's ability to develop an opportune hypersensitive response (Agrios 1997), which in turn relies on an effective and rapid signaling to induce the expression of specific defense related proteins. The activation of phosphatidylinositol4,5 bisphosphate (PIP2)-directed phospholipase -C (PLC, E. C. ) after the binding of agonist molecules to numerous mammalian cell surface receptors is known to occur, through either the action of heterotrimeric G-proteins or receptor protein tyrosine kinases. As a consequence of PLC activation, the second messengers diacylglycerol (DAG) and inositol1, 4,5triphosphate (IP3) are produced, leading to the subsequent activation of protein kinases and Ca2+ mobilization and finally to the physiological cellular response (Brearly et al, 1997; Munnik et al, 1998).

The presence of almost all the components of the phosphoinositide signaling pathway has been confirmed in many different plant systems (Munnik et al, 1998). The involvement of Ca2+ and IP3 as important second messengers in response to various stimuli has been also established (Toyoda et al, 1993; Arz and Grambow, 1995; Smolenska-Sym and Kacperska, 1996; Chrispeels et al, 1999).

Lemon seedlings inoculated with conidia of Alternaria alternata develop a hypersensitive response (HR). This is characterized by the induction of the phenylpropanoid pathway and its enzyme Phenylalanine ammonialyase (PAL, E. C. (Roco et al, 1993), the increase in umbelliferone concentration, synthesis of the phytoalexin scoparone (Pérez et al, 1994) and the expression of new chitinases and ß1,3glucanases (Ortega and Pérez, 1999). Calcium ions and calcium channels (Castañeda and Pérez, 1996) participate in early stages of transduction of the signal generated by A. alternata in lemon seedlings, but it is unknown if IP3 or internal Ca2+ mobilization are involved in this signaling event.

The use of different phosphoinositide (PI)-signaling pathway inhibitors has proven to be an effective tool for the study of a possible role of this system in mediating gene expression in other plants such as Larix decidua (Bach and Seitz, 1997), Arabidopsis thaliana (Liang et al, 1996), or Pisum sativum (Toyoda et al, 1993), among others. Similar approaches have been used to analyze internal calcium mobilization in whole plants where the use of compounds such as FURA2 is not possible due to the lack of reliable cell suspension cultures or protoplasts. We therefore decided to use these approaches to infer the participation of IP3 in the development of HR in C. limon against A. alternata.


All reagents were analytical grade and purchased from Amersham, Sigma and Merck.


Lemon seedlings were grown from seeds as described in Roco et al. (1993). A. alternata was isolated from citrus trees infected with sooty molds (Pérez et al, 1991) and their conidia obtained as recommended by AOAC (1980).

Effect of external calcium

Lemon seedlings always watered with sterile water were wounded (Roco et al, 1993) and incubated in 3 mM EGTA for 5 min prior to inoculation with fungal conidia in order to chelate external calcium ions released from the cell wall after wounding. Seedlings were processed as below to test PAL activity.

Effect of heparin, caffeine, and calcium ionophore A23187

Lemon seedlings always watered with sterile water were wounded (Roco et al, 1993) and incubated in 0 - 10 µM heparin or in 0 - 100 mM caffeine from 0 to 3 hours (controls) or for 5 minutes prior to inoculation with fungal conidia. Incubation with 10 nM A23187 for 3 hours was done as described in Castañeda and Pérez (1996). Seedlings were processed as below to test PAL activity.

Effect of neomycin

Lemon seedlings always watered with sterile water were wounded (Roco et al, 1993) and incubated in 0 - 2 mM neomycin for five minutes prior to inoculation with fungal conidia. Seedlings were processed as below to test PAL activity and scoparone synthesis.

Effect of LiCl

Lemon seedlings, either watered with a solution of LiCl (1 - 100 mM), or with NaCl (1 - 100 mM) for 16 hours before each experiment, were wounded (Roco et al, 1993). Seedlings were inoculated with fungal conidia and processed as below, to test PAL activity, synthesis of scoparone and IP3 quantitation.

To test short-term treatment with LiCl, seedlings always watered with sterile water were wounded (Roco et al, 1993) and inoculated with fungal conidia obtained in 0.15 M LiCl. Controls were inoculated with conidia obtained in 0.15 M NaCl. Seedlings were processed as below to test PAL activity and scoparone synthesis.

Plant inoculation, PAL activity and protein concentration

Inoculation was done by carefully spreading a suspension (2 x 106 conidia/mL) on top of the wounded tissue (Pérez et al, 1994). Controls were performed spreading the appropriate solution.

Homogenates from control or treated lemon seedlings were obtained as described by Roco et al, (1993). The protein concentration was estimated (Bradford, 1976), and PAL activity was assayed spectrophotometrically using L-Phe as substrate (Zucker, 1965) at different time periods depending on the elicitor used (Roco et al, 1993; Castañeda and Pérez 1996). Results are expressed as PAL ratio of treated/control seedlings or as PAL activity (pkat/mg proteins), and correspond to the mean of at least three different experiments run in duplicates. SD did not exceed 10%.

IP3 quantitation

Phosphoinositides were extracted and IP3 quantified as recommended by the manufacturer of the Amersham kit TRK 1000 (Chandok and Sopory 1994). Results are expressed as pmoles of IP3 per gram fresh weight and correspond to the mean of at least four different experiments. SD did not exceed 10%.

Analysis of the phytoalexin scoparone

Experiments were run under the same conditions used for the elicitor bioassay, with the exception that the seedlings were directly extracted with Ethyl acetate (2mL per g fresh weight of seedlings) 42 h after PAL induction (Pérez et al, 1994). The organic extract was applied to TLC plates (silica gel GF254) and developed in toluene-Ethyl acetate (1:1). The presence of scoparone was visualized under UV light (254 and 320 nm) (Alvarez and Finkelstein 1999). Scoparone concentration was estimated as described in Roco et al, (1993).


External calcium is not enough for the development of a complete hypersensitive response

The PAL activity increase observed in response to fungal conidia was reduced, but not completely suppressed by the presence of up to 3mM EGTA in these seedlings (Table I). This effect is coincident with the one observed in Larix decidua (Bach and Seitz 1997) or in Arabidopsis thaliana (Knight et al, 1997) and suggests that in addition to external calcium and calcium channels (Castañeda and Pérez 1996), internal calcium stores may also be required for a complete development of the HR. These intracellular calcium stores could probably be sensitive to the PI system stimulation as has also been observed in other plants in response to drought and salinity (Knight et al, 1997). In order to confirm this hypothesis, we decided to use different PI system inhibitors such as LiCl, which prevents PI turnover after long-term treatments (Gillaspy et al, 1995); neomycin, which inhibits binding of PLC to its substrate PIP2 (Munnik et al, 1998); heparin, which inhibits IP3 receptors (Chadwick et al, 1990); and caffeine, a known inhibitor of cAMP phosphodiesterase, which also inhibits the IP3 receptor in mammalian cells (Berridge, 1993). These tools allowed us to study the participation of IP3 in PAL induction and phytoalexin synthesis in lemon seedlings in response to Alternaria alternata.

PAL induction in response to heparin and caffeine

A two-fold increase in PAL activity was observed after treatment of lemon seedlings with 3 µM heparin (Table I). A similar effect was observed at all of the heparin concentrations tested (1 to 10 µM), suggesting either an unspecific response or a saturation of the system at 1 µM heparin. This glycosamineglucan has been described as a potent competitive inhibitor of IP3 binding to its isolated receptor. It has also been shown that heparin blocks IP3-induced intracellular calcium release in vascular smooth muscle, suggesting that the IP3 receptor regulates calcium release in mammalian systems (Chadwick et al, 1990). Nevertheless, these studies have been performed with microsomal fractions or isolated IP3 receptors for the pharmacological characterization of the latter. The chemical structure of heparin resembles that of lemon elicitors (Roco et al, 1993), and therefore the main effect observed at any of the heparin concentrations where PAL ratio is higher than one could be that of an elicitor discarding an inhibitory effect on the calcium release in this system. This result suggests that, in this system, heparin is not able to reach the IP3 receptor to exert its inhibitory effect. Moreover, PAL induction by heparin was additive to that observed in response to calcium ionophore A23187 (Table I), which is known to induce the hypersensitive response in C. limon (Castañeda and Pérez 1996).

In opposition to the expected maintenance of basal PAL activity, an increase similar to that observed for the heparin treatment was found on this enzyme activity in response to 2 mM caffeine (Table I), suggesting that the system could be responding to the sole treatment with caffeine in a non-specific way. The maximal increase in PAL activity was observed at 2 mM caffeine with 50% values at 1.3 and 4.5 mM. The induction of PAL by 2 mM caffeine was not suppressed by 3 mM EGTA, suggesting that external calcium is not involved in caffeine's ability to induce PAL. On the other hand, the increase in PAL activity in response to inoculation with fungal conidia was inhibited by 2 mM caffeine. In this situation, PAL activity corresponded to that obtained when seedlings were treated with caffeine alone (Table I). This compound, a known inhibitor of cAMP phosphodiesterase, also inhibits the IP3 receptor in mammalian systems (Berridge 1993). Therefore, caffeine may be reaching an IP3 receptor in lemon seedlings, but the possibility of other unknown effects on whole plant systems cannot be discarded. The stimulatory effects of heparin and caffeine in the absence of added fungal conidia make it difficult to use these chemicals as inhibitors of the PI signaling in whole plant systems.

PAL induction and scoparone synthesis in neomycin treated lemon seedlings

Basal PAL activity was not affected by wounding as described for our plant system, (Roco et al, 1993), nor by neomycin at concentrations between 0.25 and 2 mM. Neomycin also failed to induce the synthesis of scoparone by itself.

The PAL activity increase observed in response to the inoculation with A. alternata (Roco et al, 1993) was inhibited to a degree dependent upon the concentration of neomycin, with an estimated IC50 of 0.11 mM. Nevertheless, PAL induction was not completely suppressed by 2 mM neomycin, nor was the synthesis of scoparone (Fig. 1), confirming that any level of PAL induction observed in response to fungal inoculation results in phytoalexin synthesis in this system. A similar inhibitory effect of neomycin on PAL induction was observed for the interaction between Larix decidua and fungal cell walls from Fusarium oxysporum (Bach and Seitz, 1997) or for Pisum sativum and Micosphaerella pinodes (Toyoda et al, 1993), where 1 mM neomycin partially prevented the synthesis of the phytoalexin pisatin. Neomycin is a known PLC inhibitor (Knight et al, 1997; Munnik et al, 1998) that binds to inositol-4, 5-bisphosphate (Coté, 1995), preventing its hydrolysis (Legendre et al, 1993; Renelt et al,1993) and the resulting release of the second messenger IP3. It has been shown to be an inhibitor of IP3 production in protoplasts in response to light and elicitors (Coté 1995), in plant cell cultures (Legendre et al, 1993), or in leaves and hypocotyls (Toyoda et al, 1993) in response to elicitors. Thus, the inhibitory effect of neomycin in the development of the HR of lemon seedlings suggests that the PI signaling pathway could be involved in our plant system.

IP3 levels, PAL induction and scoparone synthesis in LiCl watered lemon seedlings

IP3 quantitation showed that seedlings had a basal second messenger content of 200 + 20 pmoles per gram fresh weight. After inoculation with A. alternata, IP3 increased 1.7-fold and 2.4-fold at 7 and 25 min, respectively (Fig 2). Basal IP3 level of C. limon agrees with that reported for other plant systems, such as P. sativum (Toyoda et al, 1993), where the basal level is of 150 pmoles per gram fresh weight. As opposed to lemon seedlings, this latter system showed only one maximum of IP3 at 7min after elicitation. During this time period, peas reached levels of 300 pmoles of IP3 per gram fresh weight, similar to levels reached in lemon seedlings for this same time period. Isolated plasma membranes from Triticum aestivum leaves treated with an elicitor from Puccinia graminis F sp. tritici (Arz and Grambow 1995) also showed a single increase in IP3 at a similar timing. However, for the pea system, the measurement of IP3 did not include time periods between 15 and 30 minutes after elicitation (Toyoda et al, 1993). Therefore, we cannot discarded the possibility that a second increase could be observed in peas, similar to the one seen in lemon seedlings at 25 minutes after elicitation. It is important to mention that a decrease of IP3 concentration below the basal level was observed at 15 minutes after elicitation both for peas (Toyoda et al, 1993) and lemon seedlings, suggesting a similar behavior of both systems once they have been elicited by the corresponding fungi. These control lemon seedlings induced PAL and formed scoparone in response to A. alternata, as described previously (Roco et al, 1993; Pérez et al, 1994).

Figure 2. Time course changes in IP3 concentration in lemon seedlings inoculated with A. alternata: effect of watering with 100 mM LiCl. IP3 was extracted and analyzed using the Amersham kit TRK 1000.

When lemon seedlings were watered long term with LiCl, the increase in IP3 production was also observed (Fig 2), but the levels reached were lower than those of controls, suggesting that Li+ affected IP3 levels after fungal inoculation. Li+, the smallest of the alkali metals, has been successfully used to investigate a possible involvement of the PI signaling pathway and calcium in mediating gene expression in other plant systems such as Arabidopsis thaliana (Liang et al, 1996; Knight et al, 1997). It is known that the action of lithium is based on its inhibitory effect on the phosphoinositide cycle. Lithium interrupts phosphoinositides cycling by non-competitively inhibiting myoinositol-1-phosphatase, the enzyme that dephosphorylates inositol-1-phosphate to yield free inositol (Gillaspy et al, 1995). This inhibitory effect leads to a reduction in the supply of inositol lipids required for IP3 production (Liang et al, 1996; Knight et al,1997). Plants do not contain lithium under normal growing conditions, but they can absorb Li+ ions from the environment or their growth medium (Liang et al, 1996). Therefore, it appears that in lemon seedlings, LiCl effectively prevented phosphoinositide turnover decreasing PIP2 concentration in the membrane without affecting the signaling system.

No PAL induction was observed in lemon seedlings inoculated with A. alternata, under the same long-term watering conditions with LiCl. The use of different LiCl watering solutions (1100 mM), allowed us to estimate an IC50 of 2mM. Seedlings that were watered with NaCl solutions (same concentrations as LiCl) acted as controls, i.e.: induced PAL in response to fungal inoculation, discarding an unspecific salt effect on the lemon system. These results agree with other reports in which none of the other alkali ions (Na+, K+, Cs+) could produce the inhibitory action of lithium (Liang et al, 1996). Finally, lemon seedlings watered over the long term with LiCl did not form scoparone (Fig. 1). Thus, it may be established that a correlation exists between IP3, PAL induction and scoparone synthesis suggesting that this second messenger is participating in the signal transduction to develop the hypersensitive response of lemon seedlings against fungal inoculation. On the other hand, and contrary to long-term watering with LiCl, short time period treatments with this cation can exert a positive effect on signal transduction as it prevents the metabolism of recently produced IP3 (Lee et al, 1996). This would result in a transitory increase in IP3 levels which would in turn increase cytosolic calcium levels (Chrispeels et al, 1999). We studied this effect using fungal conidia obtained in 150 mM LiCl or in 150 mM NaCl to elicit PAL induction in control seedlings. No differences on basal PAL activity were observed in controls, where the direct effect of these cations was tested using any of these salt solutions alone. Nevertheless, PAL induction in lemon seedlings elicited with A. alternata fungal conidia obtained in LiCl was greater than the one observed for elicitation with conidia obtained in NaCl. These results reinforce the fact that an increase in IP3 levels is necessary for PAL induction resulting in scoparone synthesis, which agrees with results from the pea system where pisatin is not found when IP3 production has been inhibited (Toyoda et al, 1993).


This work was funded by FONDECYT grants Nº 1970532 and 2990090.

Corresponding author: Luz M. Pérez. Laboratorio de Bioquímica, Facultad Ciencias de la Salud, Universidad Andrés Bello, Sazie 2325, Santiago de Chile. Telephone: 56-2-6618387. Fax: 56-2-6618390. e-mail:

Received: January 22, 2001. Accepted: March 14, 2001.


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